CN101903386A - Compounds inhibiting dipeptidyl peptidase-IV, processes for their preparation, and pharmaceutical compositions containing said compounds as active agents - Google Patents

Abstract

Disclosed herein are novel compounds of Formula (1) as defined in the specification having excellent inhibitory activity against dipeptidyl peptidase-IV (DPP-IV), methods of preparing the same and pharmaceutical compositions comprising the same as an active agent.

Description

Compounds inhibiting dipeptidyl peptidase-IV, processes for their preparation, and pharmaceutical compositions containing said compounds as active agents

technical field

The present invention relates to novel compounds having excellent inhibitory activity against dipeptidyl peptidase-IV (DPP-IV), processes for preparing said compounds, and pharmaceutical compositions containing said compounds as active agents.

Background technique

Diabetes has significant adverse effects on human health and is accompanied by various complications. There are two main types of diabetes: type 1 diabetes, characterized by little or no insulin secretion due to the destruction of pancreatic cells, and type 2 diabetes, characterized by insufficient insulin and Insulin resistance. The incidence of type II diabetes accounts for more than 90% of all diabetic patients. Representative examples of diabetic complications include hyperlipidemia, hypertension, retinopathy and renal insufficiency (Paul Zimmer et al., Nature, 2001, 414, 782). Iodonuides (stimulate pancreatic insulin secretion), biguanides (inhibit hepatic glucose production), α-glucosidase inhibitors (inhibit intestinal glucose absorption) and the like have been used as antidiabetic agents. Recently, peroxisome proliferator-activated receptor gamma (PPARγ) agonists (thiazolidinediones, which enhance insulin sensitivity) have attracted great attention as promising antidiabetic therapeutics. However, these antidiabetic drugs have adverse side effects such as hypoglycemia and weight gain (David E. Moller, Nature, 2001, 414, 821). In view of this, there is an urgent need to develop therapeutic drugs for diabetes with reduced side effects, especially those that do not cause hypoglycemia and weight gain.

At the same time, it was recently found that dipeptidyl peptidase-IV (DPP-IV) knockout (knockout) mice maintained glucagon-like protein 1 (GLP-1) activity and high insulin levels, resulting in decreased blood glucose levels, which demonstrated the therapeutic feasibility of DPP-IV as an antidiabetic agent (Marguet D. et al., Natl. Acad. Sci. USA, (2000) 97, 6874-6879). GLP-1 is involved in the differentiation and growth of pancreatic β cells in vivo, and plays an important role in the production and secretion of insulin. GLP-1 is inactivated by DPP-IV, and DPP-IV inhibitors have been reported to increase insulin secretion by inhibiting the inactivation of GLP-1. DPP-IV inhibitors have also been developed as anti-obesity drugs, as GLP-1 contributes to supersatiation in mice and slows down the digestion of food in the intestine, leading to weight loss. In addition, many researchers and institutions have also proved that in animal model experiments, DDP-IV inhibitors control blood sugar and blood lipid levels (Pospislik J.A., et al., Diabetes, (2002) 51, 943-950). In this regard, DPP-IV inhibitors may be considered as potentially useful agents for the treatment of diabetes.

To date, much research on the discovery and development of beneficial DPP-IV inhibitors has focused on materials in which the ring of pyrrolidine introduces a cyano group. For example, WO 00/34241 discloses DPP-IV inhibitors represented by the following formula.

Wherein R is a substituted adamantyl group, and n is in the range of 0-3.

Other inhibitors can be identified in WO 04/064778, WO 03/004498 and WO 03/082817, among others. DPP-IV disclosed in WO 04/064778 is a DPP-IV inhibitor represented by the following formula.

wherein Ar is unsubstituted or substituted phenyl; R ₁₅ , R ₁₆ and R ₁₇ are each independently hydrogen or alkyl; U, V and W are each independently nitrogen, oxygen or substituted nitrogen or carbon.

In addition, WO 03/004498 proposes a DPP-IV inhibitor represented by the following formula.

wherein Ar is unsubstituted or substituted phenyl; R ₁₈ is hydrogen or alkyl; and T is nitrogen or substituted carbon.

Furthermore, WO 03/082817 discloses a DDP-IV inhibitor represented by the following formula.

wherein Ar is unsubstituted or substituted phenyl; R ₁₉ , R ₂₀ and R ₂₁ are each independently hydrogen or alkyl; and Q is nitrogen or substituted carbon.

In the DPP-IV inhibitor-related patent application WO 06/104356 assigned to the applicant with the title of "DIPEPTIDYL PEPTIDASE-IVINHIBITING COMPOUNDS (dipeptidyl peptidase-IV inhibitory compounds)", the inventors have demonstrated that The compounds represented by the following formula I' have wide and comprehensive effects.

Compounds of formula I' are cyclic compounds having rings connected by means of amide bonds, similar to those DPP-IV inhibitor compounds listed in the aforementioned patent publications; however, are represented in the aforementioned documents as The molecular structure in which Ar or Z is substituted with a phenyl group is completely different from the structure in the present invention substituted with a saturated or unsaturated 5-membered or 6-membered ring moiety. Furthermore, to the best of our knowledge, the DPP-IV inhibitors of the present invention having a molecular structure in which the lactam ring is substituted at the phenyl position has not been disclosed in the art.

In addition, the inventors of the present invention have found that the compound of formula I' disclosed in the applicant's previous patent can be obtained by introducing an amine with a high DPP-IV inhibitory effect into substituent A and introducing various substituents (such as hydroxyl group) into substituent B. or carbonyl), clearly showing improved DPP-IV inhibitory activity and selectivity.

In particular, considering recent reports, the low selectivity of conventional techniques for other dipeptidyl peptidases can lead to various adverse effects (Lankas, G.R. et al., Potential Importance of Selectivity over Dipeptidyl Peptidases 8 and 9 (On Dipeptidyl Peptidases 8 and 9) The potential importance of the selectivity of enzymes 8 and 9), diabetes, 2005, 548, 2988 ~ 1994), so the present invention provides highly improved results in terms of excellent drug efficacy and high DPP-IV selectivity of the compounds of the present invention. Also, the DPP-IV inhibitor compound of the present invention having the aforementioned chemical structure and its preparation method are not disclosed in the related art.

Contents of the invention

As a result of various extensive and intensive studies and experiments in order to solve the above problems and to find compounds having excellent DPP-IV inhibitory activity and selectivity, the inventors of the present invention found that compounds having an optionally substituted lactam ring structure Effective as a DPP-IV inhibitor, as described below. The present invention has been accomplished based on these findings.

It was therefore an object of the present invention to provide DPP-IV inhibitor compounds having a lactam ring structure which is optionally substituted, especially by hydroxyl or carbonyl.

Another object of the present invention is to provide processes for the preparation of said compounds.

Another object of the present invention is to provide a pharmaceutical composition containing said compound and a method of treating or preventing a disease associated with DPP-IV, said method comprising administering an effective amount of said compound to a subject in need thereof.

According to aspects of the present invention, the above and other objects can be achieved by providing a dipeptidyl peptidase-IV (DPP-IV) inhibitor compound represented by Formula 1.

Wherein A is selected from: substituents of formula 2:

where R ₁ is hydrogen or CF ₃ ,

R ₂ is selected from hydrogen, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₃ -C ₁₀ cycloalkyl, substituted or unsubstituted C ₄ -C ₈ aryl and substituted or unsubstituted C ₃ -C ₇ heteroaryl; and

Substituents of formula 3:

Wherein R ₃ is selected from hydrogen, halogen and substituted or unsubstituted C ₁ -C ₄ alkyl;

B is selected from: substituents of formula 4:

in

x is _-CR4R5 -or-CO-, wherein _R4 and _R5 are each independently hydrogen or hydroxyl, with the proviso that _at least one of _R4 and _R5 is hydroxyl,

R ₆ , R ₇ , R ₈ and R ₉ are each independently selected from hydrogen, halogen, and substituted or unsubstituted C ₁ -C ₄ alkyl,

n is 0 or 1;

Substituents of Formula 5:

in

x is -CR ₁₀ R ₁₁ -or -CO-, wherein R ₁₀ and R ₁₁ are each independently hydrogen or hydroxyl, with the proviso that at least one of R ₁₀ and R ₁₁ is hydroxyl,

R ₁₂ , R ₁₃ and R ₁₄ are each independently selected from hydrogen, halogen, and substituted or unsubstituted C ₁ -C ₄ alkyl,

Y is oxygen or sulfur;

Substituents of formula 6:

in

x is -CR ₁₅ R ₁₆ -or -CO-, wherein R ₁₅ and R ₁₆ are each independently hydrogen or hydroxyl, with the proviso that at least one of R ₁₅ and R ₁₆ is hydroxyl,

R ₁₇ , R ₁₈ and R ₁₉ are each independently selected from hydrogen, halogen, and substituted or unsubstituted C ₁ -C ₄ alkyl,

Z is -CH- or oxygen, with the proviso that when Z is oxygen, R ₁₉ is absent; and

A substituent of formula 7;

x is -CR ₂₀ R ₂₁ -or -CO-, wherein R ₂₀ and R ₂₁ are each independently hydrogen or hydroxyl, with the proviso that at least one of R ₂₀ and R ₂₁ is hydroxyl,

R ₂₂ is substituted or unsubstituted C ₁ -C ₄ alkyl.

According to various experimental findings conducted by the inventors of the present invention, it is confirmed that the compound of formula 1 introduces an amine group having an excellent DPP-IV inhibitory effect into substituent A and introduces a specific substituent (such as hydroxyl or carbonyl) as substituent B, said compounds exhibited significantly improved DPP-IV inhibitory activity and selectivity.

Furthermore, the compounds of the present invention are capable of forming addition salts with pharmaceutically acceptable acids.

The term "pharmaceutically acceptable salt" refers to a preparation of a compound that does not cause significant irritation to the organism to which it is administered and that does not impair the biological activity and performance of the compound. Examples of pharmaceutically acceptable salts may include acid addition salts of the compounds with acids capable of forming non-toxic acid addition salts containing pharmaceutically acceptable anions, and the acids are, for example, inorganic acids such as hydrochloric acid, sulfuric acid, Nitric acid, phosphoric acid, hydrobromic acid and hydroiodic acid; organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid and salicylic acid; or sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Specifically, examples of pharmaceutically acceptable carboxylates include salts with alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, and magnesium, salts with amino acids such as lysine, arginine, and guanidine; and salts with organic bases. Such as salts of dicyclohexylamine, N-methyl-D glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline and triethylamine. The compound of formula 1 in the present invention can be converted into its salt by conventional methods known in the art.

Unless otherwise stated, the term "compound of formula 1" is intended to include the compound of formula 1 itself as well as pharmaceutically acceptable salts.

As used herein, the term "alkyl" refers to a group free of unsaturated groups and composed of carbon and hydrogen atoms. The alkyl group may be straight chain or branched. Examples of alkyl groups may include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, tert-butyl, and sec-butyl.

The lower alkyl group is a C ₁ -C ₁₀ alkyl group (for example, an alkyl group having 1 to 10 carbon atoms in a linear or branched alkyl backbone). The alkyl groups are optionally substituted. When it is substituted, the alkyl group may be substituted with four or less substituents at any bonding point (any carbon atom).

When an alkyl group is substituted by another alkyl group, it is also intended to be referred to as "branched alkyl".

As used herein, the term "cycloalkyl" refers to an alkyl species containing 3 to 15 carbon atoms, preferably 3 to 8 carbon atoms, which does not form alternating or resonant double bonds between carbon atoms. The cycloalkyl group may contain 1 to 4 rings. Examples of cycloalkyl may include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl. Exemplary substituents for cycloalkyl groups may include halogen, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkoxy, C ₁ -C ₁₀ alkylhydroxy, amino, nitro, cyano, thiol, and C ₁ -C ₁₀ alkylthio.

Heterocycloalkyl is a saturated or unsaturated 7-11 membered bicyclic heterocyclic ring system or a stable non-aromatic 3-8 membered monocyclic heterocyclic ring system, or it may be a fused ring, spiro ring or bridged ring system , in which ring carbons are replaced by heteroatoms such as nitrogen (N), sulfur (S) or oxygen (O). Each heterocyclic ring consists of one or more carbon atoms and 1 to 4 heteroatoms. A heterocycloalkyl group can be attached to any endocyclic carbon atom that renders the structure stable. Preferred examples of heterocyclic groups may include, but are not limited to, furan, thiophene, pyrrole, pyrroline, pyrrolidine, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, iso Thiazole, triazole, thiadiazole, pyran, pyridine, piperidine, morpholine, thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine and triazine. Examples of heterocycles may include, but are not limited to, 3-7 membered monocyclic heterocycles such as piperidinyl, pyranyl, piperazinyl, morpholinyl, thiomorpholinyl and tetrahydrofuryl, more preferably 3-7 membered monocyclic heterocycle.

The term "aryl" refers to an aromatic group having at least one ring with a conjugated pi-electron system, aryl includes carbocyclic aryl (eg, phenyl) and heterocyclic aryl (eg, pyridine). The term is intended to include monocyclic or fused-ring polycyclic (ie, rings that share adjacent pairs of carbon atoms) groups. The aryl group may optionally contain 1 to 4 heteroatoms (such as nitrogen (N), sulfur (S) or oxygen (O)) in the carbocyclic and aromatic rings, and such heteroatom-containing aryl systems are also referred to as "heteroaryl".

Examples of aryl or heteroaryl groups may include, but are not limited to, phenyl, naphthyl, pyridyl, pyrimidinyl, pyrrolyl, isothiazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isothiazolyl, Oxazolyl, pyrazinyl, pyridazinyl, triazinyl, quinazolinyl, thiazolyl, benzothienyl, furyl, imidazolyl and thienyl.

In the compound of formula 1 of the present invention, when the alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is substituted, the substituent can be C ₁ -C ₁₀ alkyl or halogen, especially preferably fluorine Substituted C ₁ -C ₁₀ alkyl.

In a preferred embodiment of the present invention, A in formula 1 is a substituent shown in formula 2, wherein R ₁ is hydrogen or CF ₃ , and R ₂ is selected from hydrogen, and C ₁ -C ₅ alkyl, C ₃ -C ₆ cycloalkyl, C ₃ -C ₁₀ heterocycloalkyl, C ₄ -C ₈ aryl and C ₃ -C ₇ heteroaryl, each of which may be optionally substituted by halogen.

When R ₂ is unsubstituted or halogen-substituted C ₃ -C ₁₀ heterocycloalkyl, or unsubstituted or halogen-substituted C ₃ -C ₇ heteroaryl, then the heterocycloalkyl or heteroaryl may be Any one selected from the following: furan, thiophene, pyrrole, pyrrolidine, imidazole, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, iso Thiazolidine, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran , dihydropyran, tetrahydropyran, pyridine, pyridone, pyridazine, pyrazine, pyrimidine, piperidine, piperazine, morpholine, pyridazinone, tetrazole, triazole, triazolidine and azepine.

In a more preferred embodiment of the invention, R _can be selected from trifluoromethyl, propyl, butyl, tert-butyl, cyclobutyl, pyridine, furan, methoxyethyl, thiophene and 4-fluorobenzene base.

In addition, B in Formula 1 is a substituent represented by Formula 4, wherein X is -(CH-OH)- or -CO-, R ₆ and R ₇ are each independently selected from hydrogen, fluorine and unsubstituted C ₁ -C ₄ alkyl, and R ₈ and R ₉ are each independently hydrogen.

The compounds of the present invention include their isomers. Compounds in which the NH ₂ substituted carbon atom forms a stereogenic center are especially preferred, as shown in the structure of formula 1a.

wherein A and B are as defined in Formula 1.

Particularly preferred, non-limiting examples of compounds of Formula 1 of the present invention may include the following compounds:

1-[(2S)-amino-4-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-4- Oxygen-butyl]-5,5-difluoro-(6R)-hydroxyl-piperidin-2-one,

1-[(2S)-amino-4-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-4- Oxygen-butyl]-5,5-difluoro-(6S)-hydroxy-piperidin-2-one,

1-[(S)-2-amino-4-oxo-4-(2-propyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine- 7-yl)-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-amino-4-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl )-4-oxo-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-amino-4-(2-cyclobutyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl )-4-oxo-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-amino-4-oxo-4-(2-pyridin-4-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-amino-4-oxo-4-(2-pyridin-4-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-amino-4-(2-furan-2-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine-7 -yl)-butyl]-4-oxo-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one,

1-[(S)-2-amino-4-(2-furan-2-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine-7 -yl)-butyl]-4-oxo-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one,

1-{(S)-2-amino-4-[2-(2-methoxy-ethyl)-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4- d] pyrimidin-7-yl]-4-oxo-butyl}-5,5-difluoro-6-hydroxyl-piperidin-2-one,

1-{(S)-2-amino-4-[2-(2-methoxy-ethyl)-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4- d] pyrimidin-7-yl]-4-oxo-butyl}-5,5-difluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-Amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-Amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one,

1-{(S)-2-amino-4-[2-(4-fluoro-phenyl)-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d] Pyrimidin-7-yl]-4-oxo-butyl}-5,5-difluoro-6-hydroxy-piperidin-2-one,

1-[(S)-2-amino-4-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)- 4-oxo-butyl]-3,3-difluoro-piperidine-2,6-dione,

1-[(S)-2-amino-4-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl )-4-oxo-butyl]-5-fluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-amino-4-(2-furan-2-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine-7 -yl)-4-oxo-butyl]-5-fluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-Amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5-fluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-amino-4-oxo-4-(2-propyl-5-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine- 7-yl)-butyl]-5-fluoro-6-hydroxy-piperidin-2-one,

1-[(S)-2-amino-4-oxo-4-(2-pyridin-4-yl-5-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5-fluoro-6-hydroxyl-piperidin-2-one,

1-[(S)-2-Amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-3-methyl-piperidine-2,6-dione, and

1-[(S)-2-amino-4-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl )-4-oxo-butyl]-3-methyl-pyrrolidine-2,6-dione.

The compound of the present invention or a pharmaceutically acceptable salt thereof may be in the form of a hydrate or a solvate.

In addition, the present invention provides methods for preparing compounds of formula 1 .

An exemplary method of preparing a compound of Formula 1 includes reacting a compound of Formula 8 with a compound of Formula 9 and removing the amine protecting group _P1 .

AH ₂ F ₁ (9)

In Equation 8 and Equation 9,

A and B are as defined above;

P ₁ is an amine protecting group; and

F ₁ is absent or hydrochloric acid, sulfuric acid or trifluoroacetic acid.

Reaction Scheme 1

In Reaction Scheme 1, A, B and _P1 are as defined above; and _G1H is hydrochloric acid, sulfuric acid or trifluoroacetic acid.

Reagents and Reaction Conditions

a: EDCI, HOBT, _Et3N or _AH2G1H

b: G ₁ H

Specifically, Compound 1 can be obtained as follows. First, the desired amine group is introduced into the compound of formula 8 by means of a coupling reaction using EDC and HOBT, thereby forming compound 10 as an amide. Then, use a strong acid such as TFA or HCl when the amine protecting group _P1 is Boc, or use _H2 /Pd/C or TMSI when the amine protecting group _P1 is Cbz, or when the amine protecting group _P1 is Fmoc The amine protecting group P1 was removed using _Et2NH to afford compound 1.

In reaction step a), amine A can be obtained by using the methods disclosed in WO 04/064778, WO 03/004498, WO 03/082817, WO 06/104356, etc., or can otherwise be commercially available of amines.

Meanwhile, the compound of formula 8 can be typically prepared by the following reaction scheme 2.

Reaction Schema 2

In Reaction Scheme 2, B and _P1 are as defined above; and R is phenyl, methyl, ethyl or isopropyl.

Reagents and Reaction Conditions

a: ClCO ₂ i-Bu, NMM, THF; NaBH ₄ or H ₂ O

b: BH, DIAD, PPh ₃ or THF

c: NaBH ₄ , MeOH or SiO ₂ column chromatography

d: Pd/C, H ₂ (phenyl ester), LiOH-H ₂ O or MeOH-H ₂ O (methyl, ethyl or isopropyl ester)

Specifically, the carboxylic acid of formula 11 is converted into anhydrous ester, and then the ester compound is reduced using sodium borohydride ((NaBH ₄ ), thereby obtaining compound 12 as a primary alcohol. Using DIAD and PPh ₃ can obtain The resulting coupling product of the primary alcohol and the desired imide compound, compound 13. Then, use a strong acid such as TFA or HCl when the amine protecting group _P is Boc, or use a strong acid such as TFA or HCl when the amine protecting group _P is Cbz Removal of amine protecting group P1 using _H2 /Pd/C or TMSI, or _Et2NH when amine protecting group _P1 is Fmoc affords compound 8.

Meanwhile, the substituent B of Formula 8 can be typically prepared according to the following Reaction Scheme 3-1 or Reaction Scheme 3-2.

Reaction scheme 3-1

In Reaction Scheme 3-1, Ra and Rb are each independently hydrogen or halogen.

Reagents and Reaction Conditions

a: ethyl acrylate, Cu powder, TMEDA, THF

b: MeOH with _NH3

c: NaOEt, EtOH; HCl

Specifically, the diester compound of the formula 14 is obtained by means of the Michael addition reaction of the compound of the formula 14 and ethyl acrylate, and the compound of the formula 16 containing an amide group is obtained by reacting the obtained diester with ammonia. compound of. It can be seen that, in both esters, the reaction proceeds towards the carbonyl group adjacent to the halogen due to the electron attraction of the halogen atom(s) contained in the reacting compound. Finally, cyclization in ethanol in the presence of sodium ethoxide affords the salt compound with sodium attached to the nitrogen atom of the imide. Treatment of the salt compound with anhydrous strong acid provides the desired imide compound of formula 17. The resulting insoluble NaCl solid was removed by brief filtration.

Reaction scheme 3-2

In Reaction Scheme 3-2, n is 0 or 1; R' is methyl or hydrogen; R" is hydroxyl or amino; and Rc is C ₁ -C ₄ alkyl.

Reagents and Reaction Conditions

a: Ac ₂ O

b: _NH4OH , THF

c: Ac ₂ O

If R' is methyl, the product compound is hydrolyzed by reaction with a strong base such as lithium or potassium hydroxide prior to use.

Acetic anhydride is added as a solvent and reactant to the diacid compound of formula 18 to form a highly reactive intermediate in the form of an anhydride ring. Compounds of formula 20 are generated by ring opening of the intermediate with ammonia. Since there is no strong electron-attracting substituent unlike Reaction Scheme 3-1, ammonia can attack the carbon atoms of the two ketones, and thus two types of amino acid compounds are produced. These amino acid compounds are not detrimental to subsequent reactions, and thus can be used directly without further isolation. Finally, the desired imide compound of formula 21 can be obtained by cyclization of compound 20 in amino acid form by addition of acetic anhydride as shown in step (a).

Unless otherwise stated, the starting compounds used in the present invention are known in the art, or can be synthesized from known compounds by methods known per se or methods analogous thereto.

The compound of formula 1 can be separated and purified from the reaction product by conventional methods such as recrystallization, ion electrophoresis, silica gel column chromatography or ion exchange resin chromatography.

As mentioned above, the compounds of the present invention, the starting materials for the preparation of said compounds and intermediates can be synthesized by various methods, and therefore, with respect to the preparation of the compound of formula 1, those methods should be considered to be included in the scope of the present invention Inside.

In addition, the present invention provides a pharmaceutical composition for inhibiting DPP-IV, which comprises the compound of formula 1 or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition may contain the compound of the present invention and other chemical components such as diluents, carriers and the like. Thus, if desired, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier, diluent or excipient, or any combination thereof. The pharmaceutical composition facilitates in vivo administration of the compound to the subject organism. Various techniques of administering the compounds are known in the art, including but not limited to oral, injection, aerosol, parenteral and topical administration.

The term "carrier" refers to a chemical compound that facilitates the incorporation of the compound into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly utilized carrier because it facilitates the uptake of many organic compounds into the cells or tissues of an organism.

The term "diluent" refers to a chemical compound diluted in water that dissolves the composition of interest and stabilizes the biologically active form of the composition. Salts dissolved in buffers are used as diluents in the art. One commonly used buffer is phosphate buffered saline (PBS) because it mimics the saline conditions of body fluids. Because buffer salts are able to control the pH of a solution at low concentrations, buffer diluents rarely alter the biological activity of a compound.

The term "physiologically acceptable" refers to a carrier or diluent that does not impair the biological activity and physical properties of the compound.

According to the intended use, the compounds of the present invention can be formulated into various pharmaceutical dosage forms. In the preparation of the pharmaceutical composition of the present invention, the active agent, more specifically the compound of formula 1 or a pharmaceutically acceptable salt thereof, is mixed with one or more pharmaceutically acceptable carriers, and the carrier is selected according to the prepared dosage form . For example, the pharmaceutical composition of the present invention can be formulated into dosage forms suitable for injection or oral administration.

The compounds of the present invention may be formulated in conventional manner using known pharmaceutically acceptable carriers and excipients, and presented in unit dosage form or in multi-dose containers. The formulations may take such forms as solutions, suspensions or emulsions in oily or aqueous vehicles, and may contain conventional dispersing, suspending or stabilizing agents. Alternatively, the active agent may be in powder form for reconstitution with sterile, pyrogen-free water before use. The compounds of this invention may also be formulated as suppositories containing conventional suppository bases such as cocoa butter or other glycerides. Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. Preferred dosage forms are capsules and tablets. Tablets and pills are preferably coated. By mixing the compound of the present invention as an active agent with one or more inert diluents such as sucrose, lactose, starch, etc. and carriers such as lubricants (such as magnesium stearate), disintegrants, binders, etc., oral Solid dosage form for administration.

The compounds of the invention and compositions containing the compounds can be administered with other pharmaceutical agents, such as other antidiabetic agents, if desired.

When the formulation is presented in unit dosage form, the compound of formula 1 as the active agent preferably comprises a unit dosage of about 0.1 to 1500 mg. The dosage of the compound of formula 1 will depend on the weight and age of the subject, the nature and severity of the affliction, and the judgment of the prescribing physician. For adult administration, the required dose is 1 to 500 mg/day depending on the frequency and intensity of the dose. For intramuscular or intravenous administration in adults, a total dose of about 5 to 300 mg will be sufficient. In some patients the daily dose will be higher than this.

By using an effective amount of a compound of formula 1, the present invention provides methods of treating or preventing diseases involving inappropriate activity of DPP-IV. Representative examples of diseases associated with DPP-IV include, but are by no means limited to, diabetes, obesity, etc. as described above. Among them, the present invention is preferably directed to the treatment and prevention of type II diabetes. The term "treating" means that a compound of Formula 1 or a composition containing the compound, when administered to a subject exhibiting symptoms of a disease, terminates or delays the progression of the disease. The term "prevention" refers to terminating or delaying the symptoms of a disease when a compound of formula 1 or a composition containing the compound is administered to a subject who does not exhibit disease symptoms, but is at high risk of developing disease symptoms.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Now, the present invention will be described in more detail with reference to the following examples. These examples are provided only to illustrate the present invention and should not be construed as limiting the scope and spirit of the present invention.

Preparation Example 1: Synthesis of 2,4-bis-trifluoromethyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine salt salt

(1) Synthesis of tert-butyl 3-hydroxy-piperidine-1-carboxylate

1.0 equivalent of dibutyl dicarbonate (159 g) in 200 mL of dichloromethane was slowly added to 3-hydroxypiperidine hydrochloride (100 g, 0.73 mol) and 1.1 equivalent of triethylamine (111 mL) in dichloromethane at room temperature. in a mixture of methyl chloride (800 mL). The resulting mixture was then stirred for an additional 2 hours. After the reaction was completed, the reaction solution was washed with 1.0N aqueous hydrochloric acid (1.0 L), and the organic layer was concentrated to obtain 138 g (yield: 94%) of the title compound as a white solid.

¹ H NMR (500MHz, CDCl ₃ ) δ1.35-1.55 (11H, m), 1.75 (1H, m), 1.88 (1H, m), 3.08 (2H, m), 3.53 (1H, m), 3.73 ( 2H, br d, J = 5.6 Hz).

(2) Synthesis of tert-butyl 3-oxo-piperidine-1-carboxylate

Dissolve tert-butyl 3-hydroxy-piperidine-1-carboxylate (4.0 g, 20 mmol) synthesized in section (1) in a mixed solution of toluene, water and ethyl ester (2:1:2, 100 mL) , and 1.0 mol% TEMPO (31 mg) and NaBr (2.3 g) were sequentially added thereto. To the mixture was slowly added a mixture of NaHCO ₃ (4.7 g) and NaOCl (5%, 36 mL) under cooling (0˜4° C.). After the reaction was complete, the organic layer was dried over anhydrous magnesium sulfate and filtered. The filtered organic layer was concentrated under reduced pressure to obtain 3.8 g (yield: 95%) of the title compound.

¹ H NMR (500MHz, CDCl ₃ ) δ1.47(s, 9H), 1.98(m, 2H), 2.47(t, J=6.7Hz, 2H), 3.59(t, J=6.1Hz, 2H), 4.00 (bs, 2H).

(3) Synthesis of tert-butyl 3-oxo-4-(2,2,2-trichloroacetyl)-piperidine-1-carboxylate

The tert-butyl 3-oxo-piperidine-1-carboxylate (7.7g, 38.4mmol) synthesized in section (2) was dissolved in anhydrous THF (60mL) and cooled (-10～-5 ° C), the solution thus obtained was added to 1.0 M LHMDS (46 mL) while stirring, followed by stirring for 30 minutes. Under the same cooling condition, ethyl trifluoroacetate (6.6 g) was added dropwise to the mixture. The reaction was slowly warmed to room temperature and quenched by the addition of saturated aqueous acidic solution (100 mL). The reaction solution was extracted twice with ethyl acetate (100 mL), dried over anhydrous magnesium sulfate, and filtered. The extract was distilled under reduced pressure, thereby obtaining 9.5 g (yield: 80%) of the title compound.

¹ H NMR (500 MHz, CDCl ₃ ) δ 1.48 (s, 9H), 2.58 (bt, 2H), 3.57 (t, J=5.8 Hz, 2H), 4.23 (bs, 2H).

(4) Synthesis of 2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine-7(8H)- tert-butyl carboxylate

At 80° C., tert-butyl 3-oxo-4-(2,2,2-trichloroacetyl)-piperidine-1-carboxylate (2.96 g, 10 mmol) synthesized in section (3) Dissolve in pyridine (10 mL) and add it slowly to acetidine (2.0 g) while stirring. After the reaction was complete, pyridine was removed by distillation under reduced pressure. The obtained concentrate was dissolved in dichloromethane and washed with 1.0N aqueous hydrochloric acid. The organic layer was distilled under reduced pressure to obtain 3.24 g of the title compound.

¹ H NMR (500 MHz, CDCl ₃ ) δ 1.50 (s, 9H), 3.12 (bt, 2H), 3.78 (t, J=5.8 Hz, 2H), 4.85 (s, 2H).

(5) Synthesis of 2,4-bis-trifluoromethyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine hydrochloride

2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine-7(8H)-carboxylic acid tert-butyl ester synthesized in section (4) (7.5 g, 20 mmol) was dissolved in 100 mL of ethyl acetate which had been saturated with hydrochloric acid gas, and the mixture was stirred at room temperature for 1 hr. The solvent and remaining hydrochloric acid gas were distilled off under reduced pressure, and the resulting brown solid was slurried with tert-butyl methyl ether for 1 hour. The residue was filtered to obtain 5.1 g of the title compound.

¹ H NMR (400MHz, CD ₃ OD) δ 3.41(t, J=6.4Hz, 2H), 3.68(t, J=6.4Hz, 2H), 4.66(s, 2H).

Preparation Example 2: Synthesis of 2,4-bis-trifluoromethyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine salt salt derivative biology

Similar to the procedure of Preparation Example 1, the following derivatives were synthesized.

(1) 2-Propyl-4-trifluoromethyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine hydrochloride

(2) 2-tert-butyl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidine hydrochloride

(3) 2-Cyclobutyl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidine hydrochloride

(4) 2-pyridin-4-yl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidine hydrochloride

(5) 2-furan-2-yl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidine hydrochloride

(6) 2-(2-Methoxy-ethyl)-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidine hydrochloride

(7) 2-thiophen-3-yl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3,4-d]pyrimidine hydrochloride

(8) 2-(4-fluoro-phenyl)-4-trifluoromethyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine hydrochloride

Preparation Example 3: Synthesis of 3,3-difluoropiperidine-2,6-dione

(1) Synthesis of 2,2-difluoroglutarate diethyl ester

Commercially available ethyl bromodifluoroacetate (16 mL, 120 mmol) and ethyl acrylate (11 mL, 100 mmol) were dissolved in tetrahydrofuran (100 mL), and copper powder (15.2 g, 240 mmol) was sequentially added thereto at room temperature and TMEDA (16.7 mL, 110 mmol). After stirring for about 8 hours, the reaction was quenched by adding a saturated aqueous solution of 1.0 N hydrochloric acid, and extracted twice with toluene. The reaction solution was distilled under reduced pressure, and the concentrated title compound was used directly in the subsequent step without further purification.

¹ H NMR (500MHz, CDCl ₃ ) δ1.25(t, J=7.2Hz, 3H), 1.35(t, J=7.2Hz, 3H), 2.41(m, 2H), 2.52(m, 2H), 4.15 (q, J=7.2Hz, 2H), 4.32(q, J=7.2Hz, 2H).

(2) Synthesis of ethyl 4-carbamoyl-4,4-difluorobutyrate

Diethyl 2,2-difluoroglutarate (21 g) synthesized in section (1) was dissolved in ethanol (50 mL), and 2.0 M methanolic ammonia was slowly added thereto. The mixture was stirred at room temperature for 30 minutes and distilled under reduced pressure to give 18.2 g of the title compound as a viscous oil which was used in the subsequent step without further purification.

¹ H NMR (500MHz, CDCl ₃ ) δ1.25(t, J=7.2Hz, 3H), 2.43(m, 2H), 2.54(m, 2H), 4.14(q, J=7.2Hz, 2H), 5.92 (br s, 1H), 6.30 (br s, 1H).

(3) Synthesis of 3,3-difluoropiperidine-2,6-dione

Dissolve ethyl 4-carbamoyl-4,4-difluorobutyrate (18 g) synthesized in Section (2) in ethanol (100 mL), and slowly add a 1.0 N ethanol solution of sodium ethoxide (105 mL ). After stirring at room temperature for 3 hours, the pH of the mixture was adjusted to 3-4 by adding 3.0N hydrochloric acid in dioxane, and the generated NaCl was removed by filtration. The high viscosity oil was slurried with diethyl ether (100 mL). The resulting white solid was filtered and dried to obtain 11.4 g of the title compound.

¹ H NMR (500MHz, DMSO-d ₆ ) δ2.56(m, 2H), 2.71(t, J=6.8Hz, 2H), 11.60(br s, 1H)

Preparation Example 4: Synthesis of (3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2-hydroxyl-6-oxo- piperidin-1-yl)-butyric acid

(1) Synthesis of (3S)-tert-butoxycarbonylamino-4-hydroxybutyric acid isopropyl ester

Commercially available (2S)-tert-butoxycarbonylamino-4-isopropyl succinate (2.8 g, 10.3 mmol) was dissolved in tetrahydrofuran (10 mL), and N-methylmorpholine (1.13 mL). The mixture was cooled to -10°C. Isobutyl chloroformate (1.40 mL) was added to the mixture, and the mixture was stirred for 10 minutes to form an active ester. After the generated ammonium hydrochloride salt was removed by filtration, water containing NaBH ₄ (0.59 g) was added dropwise to the filtrate, and the reaction was quenched by adding saturated NH ₄ Cl. The resulting reaction slurry was extracted twice with ethyl acetate, and the organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was distilled under reduced pressure to obtain 2.51 g (yield: 95%) of the title compound as a pale yellow oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ1.22 (d, J=6.8, 6H), 1.44 (s, 9H), 2.54-2.62 (m, 2H), 3.65 (d, J=4.0, 2H), 3.99 (bs 1H), 5.02 (m, 1H), 5.36 (bs, 1H).

(2) Synthesis of (3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2,6-dioxo-piperidin-1-yl)- Isopropyl Butyrate

(3S)-tert-butoxycarbonylamino-4-hydroxybutyric acid isopropyl ester (5.3g, 20mmol) synthesized in Section (1) and 3,3-difluoropiperidine synthesized in Preparation Example 3- 2,6-Diketone (3.6 g, 26 mmol) was dissolved in dichloromethane (20 mL) and cooled to a temperature of 0-5°C. Triphenylphosphine (6.3 g) and toluene containing 2.2 MDEAD (11 mL) were added sequentially, and the reaction temperature was slowly raised to room temperature. After stirring for 4 hours, the reactant was directly subjected to column chromatography without a special termination step, whereby 5.5 g (yield: 70%) of the title compound were obtained.

¹ H NMR (400MHz, CDCl ₃ ) δ1.22(m, 6H), 1.35(s, 9H), 2.33-2.56(m, 4H), 2.87(m, 2H), 3.70(br d, J=11.8, 1H), 4.07-4.19 (m, 3H), 5.04 (m, 1H), 5.11 (br d, J=9.8, 1H).

(3) Synthesis of (3S)-tert-butoxycarbonyl-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-yl)- Isopropyl Butyrate

(3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2,6-dioxo-piperidin-1-yl)-butyric acid isopropyl ester synthesized in section (2) (5.5g, 14mmol) was dissolved in a mixed solvent of tetrahydrofuran and methanol (2:1, 60mL), and cooled to a temperature of 0-5°C. While maintaining the cooling temperature, 1.0 equiv of NaBH ₄ (530 mg) was added, and the mixture was stirred for 30 minutes. The reaction was quenched by adding saturated aqueous NH ₄ Cl solution, and purified by column chromatography to obtain 4.0 g of the title compound.

¹ H NMR (500MHz, CDCl ₃ ) δ1.22(m, 6H), 1.41(br s, 9H), 2.15(m, 1H), 2.45-2.70(m, 5H), 3.05(m, 0.4H), 3.42(m, 0.6H), 3.80(m, 0.6H), 4.10-4.35(m, 2.4H), 4.88-5.10(m, 2H), 5.35-5.60(m, 1.6H), 6.15(br s, 0.4H)

(4) Synthesis of (3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidine-1- base)-butyric acid

(3S)-tert-butoxycarbonyl-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-yl)-butyric acid isopropyl ester synthesized in section (3) (4.0 g, 10 mmol) was dissolved in a mixed solvent of tetrahydrofuran, H ₂ O and methanol (1:1:1, 30 mL), and lithium hydroxide hydrate (630 mg) was added thereto at room temperature. After 2 hours, the reaction solution was diluted with 100 mL of distilled water, and impurities were washed with dichloromethane (50 mL). The pH of the basic aqueous solution was adjusted to 3.0-4.0 by adding a strong acidic aqueous solution, and the resulting white slurry was extracted twice with dichloromethane. The organic extract was distilled under reduced pressure to provide 1.9 g of the concentrated title compound which was used in the subsequent step without further purification.

¹ H NMR (500MHz, DMSO-d ₆ ) δ1.31(br s, 9H), 2.05(m, 1H), 2.20-2.50(m, 5H), 2.85(m, 0.6H), 3.05(m, 0.4 H), 3.60(m, 2H), 3.98(m, 1), 4.80(m, 1H), 6.50(br s, 0.4H), 6.75(br s, 0.6H), 7.02(m, 1H), 12.05 (br s, 1H)

Preparation Example 5: Synthesis of [3(S)-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d] Pyrimidin-7-yl)-1-(3,3-difluoro-2-hydroxyl-6-oxo-pyridin-1-ylmethyl)-3-oxo-propyl]-aminomethyl sour tert Butyl ester

(3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-yl)butyric acid (2.6g, 4mmol) synthesized in Preparation Example 4 ) and 2,4-bis-trifluoromethyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine hydrochloride (1.4g) synthesized in Preparation Example 1 were dissolved in dichloro methane (50 mL) and cooled to a temperature of 0-4°C. HOBT (650 mg), diisopropylethylamine (2.2 mL) and EDCI (1.20 g) were sequentially added thereto, and the reaction temperature was slowly raised to room temperature. After stirring for 6 hours, the reaction solution was washed with a 1.0N aqueous hydrochloric acid solution, and the organic layer was distilled under reduced pressure to obtain 2.6 g of the title compound.

¹ H NMR (500MHz, CDCl ₃ ) δ1.31(m, 9H), 2.14(m, 1H), 2.30-2.85(m, 3H), 2.90-3.30(m, 3H), 3.45(m, 0.5H) , 3.60-4.30(m, 3H), 4.75-5.05(m, 2H), 5.95-6.50(m, 1H)

Preparation Example 6: Synthesis of [(S)-1-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-ylmethyl)-3-oxo -(2-Propyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-propyl]amino tert-butyl formate

(3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2hydroxy-6-oxo-piperidin-1-yl)-butyric acid (20mg, 0.06mmol) synthesized in Preparation Example 4 ) and 2-propyl-4-trifluoromethyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine hydrochloride (22mg) synthesized in Preparation Example 2-(1) Dissolve in dimethylformaldehyde (2 mL) and cool to a temperature of 0-4°C. HOBT (650 mg), diisopropylethylamine (2.2 mL) and EDCI (1.20 g) were sequentially added thereto, and the reaction temperature was slowly raised to room temperature. After the reaction solution was stirred for 10 hours, an aqueous ammonium chloride solution was added, and the organic layer was extracted with ethyl acetate. The residue was purified by prep-TLC to obtain 7.4 mg (yield: 22%) of the title compound.

¹ H NMR (400MHz, CDCl ₃ ) δ0.98-1.02(m, 3H), 1.26-1.30(m, 2H), 1.40-1.46(m, 9H), 1.75-1.86(m, 3H), 2.17(m , 1H), 2.55-2.69(m, 3H), 2.88-3.10(m, 5H), 3.49-4.31(m, 4H), 4.70-4.96(m, 3H) MS(m/e)602(M+Na )

Example 1: Synthesis of 1-[(2S)-amino-4-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyridine And[3,4-d]pyrimidin-7-yl)-4-oxo-butyl]-5,5-difluoro-(6R)-hydroxy-piperidin-2-one hydrochloride

[3(S)-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-1 synthesized in Preparation Example 5 -(3,3-Difluoro-2-hydroxy-6-oxo-pyridin-1-ylmethyl)-3-oxo-propyl]-carbamic acid tert-butyl ester (2.0 g) as a mixture of diastereomers, It was then separated into two isomers by silica gel column chromatography. Apart from the separated isomers, the isomer with relatively high polarity was dissolved in ethyl acetate (10 mL), treated with commercially available 4.0 N hydrogen chloride in dioxane, and stirred for 30 minutes. The reaction solution was distilled under reduced pressure, and the viscous oil was slurried with tert-butyl methyl ether (10 mL). The resulting slurry was filtered and dried to obtain 500 mg of the title compound.

¹ H NMR (500MHz, DMSO-d ₆ ) δ2.16(m, 1H), 2.35-2.55(m, 2H), 2.78-2.88(m, 2H), 2.95-3.18(m, 2H), 3.40(m , 1H), 3.70(m, 1H), 3.82(m, 2H), 4.90(m, 2H), 7.30(br s, 1H), 7.95(m, 3H)

Example 2: Synthesis of 1-[(2S)-amino-4-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyridine And[3,4-d]pyrimidin-7-yl)-4-oxo-butyl]-5,5-difluoro-(6S)-hydroxy-piperidin-2-one hydrochloride

Similar to Example 1, the diastereomeric mixture synthesized in Preparation Example 5 was subjected to column chromatography to separate relatively less polar isomers, which were then dissolved in ethyl acetate (10 mL) , then treated with commercially available 4.0N hydrogen chloride in dioxane and stirred for 30 minutes. The reaction solution was distilled under reduced pressure, and the obtained concentrate was slurried with tert-butyl methyl ether (10 mL). The resulting slurry was filtered and dried to obtain 250 mg of the title compound.

¹ H NMR (500MHz, DMSO-d ₆ ) δ2.13(m, 1H), 2.35-2.55(m, 2H), 2.75-2.90(m, 2H), 2.98(br s, 1H), 3.12(br s , 1H), 3.50-3.74(m, 2H), 3.80(m, 3H), 3.82(m, 2H), 4.90(m, 2H), 7.25(br s, 1H), 7.95(m, 3H)

Example 3: Synthesis of 1-[(S)-2-amino-4-oxo-4-(2-propyl-4-trifluoromethyl-5,8-dihydro -6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one hydrochloride

The compound synthesized in Preparation Example 6 was treated with a commercially available 4.0N solution of hydrogen chloride in dioxane and stirred for 1 hour. The reaction solution was distilled under reduced pressure, and purified by prep-TLC, thereby obtaining 4.7 mg (yield: 77%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ0.87-0.91(m, 3H), 1.72-1.80(m, 3H), 2.45-2.64(m, 6H), 2.81-3.06(m, 4H), 3.25 -3.80(m, 5H), 4.19-4.78(m, 3H); MS(m/e) 480(M+1)

Preparation Example 7: Synthesis of [(S)-3-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido [3,4-d]pyrimidin-7-yl)-1-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-ylmethyl)-3-oxo-propyl]- tert-butyl carbamate

Similar to the procedure of Preparation Example 6, (3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-yl synthesized in Preparation Example 4 )-butyric acid (26mg, 0.07mmol) and 2-tert-butyl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido [3, 4-d] Pyrimidine hydrochloride (30 mg) was coupled to obtain 15.5 mg (yield: 36%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.38-1.42(m, 18H), 2.16-2.24(m, 2H), 2.58-2.72(m, 5H), 2.99-3.12(m, 3H), 3.81 -3.89(m, 3H), 4.30(m, 1H), 4.74-4.97(m, 3H); MS(m/e) 616(M+Na)

Example 4: Synthesis of 1-[(S)-2-amino-4-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro-6H- Pyril Pyrido[3,4-d]pyrimidin-7-yl)-4-oxo-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one hydrochloride

Similar to the procedure of Example 3, 10.5 mg (yield: 76%) of the title compound was obtained using the compound synthesized in Preparation Example 7.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.30(d, 9H), 2.07(b, 1H), 2.42-2.56(m, 4H), 2.61-2.98(m, 3H), 3.21-3.30(m , 2H), 3.70-3.80(m, 3H), 4.69-4.79(m, 3H); MS(m/e) 494(M+1)

Preparation Example 8: Synthesis of [(S)-3-(2-cyclo-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d] Pyrimidin-7-yl)-1-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-ylmethyl)-3-oxo-propyl]-aminomethyl tert-butyl acid

Similar to the procedure of Preparation Example 6, (3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-yl synthesized in Preparation Example 4 )-butyric acid (26mg, 0.07mmol) and 2-cyclobutyl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3, 4-d] Pyrimidine hydrochloride (30 mg) was coupled to obtain 14.6 mg (yield: 34%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.39(m, 9H), 1.96-2.24(m, 4H), 2.38-2.74(m, 9H), 2.99-3.12(m, 3H), 3.82-3.89 (m, 4H), 4.30(m, 1H), 4.78-4.97(m, 3H); MS(m/e) 614(M+Na)

Example 5: Synthesis of 1-[(S)-2-amino-4-(2-cyclobutyl-4-trifluoromethyl-5,8-dihydro-6H- Pyrido[3,4-d]pyrimidin-7-yl)-4-oxo-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one hydrochloride

Similar to the procedure of Example 3, 13 mg of the title compound was quantitatively obtained using the compound of Preparation Example 8.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.87-2.02(m, 3H), 2.26-2.52(m, 9H), 2.89-2.99(m, 2H), 3.21-3.25(m, 2H), 3.70 -3.80(m, 4H), 3.99-4.80(m, 3H); MS(m/e) 492(M+1)

Preparation Example 9: Synthesis of [(S)-1-(3,3-difluoro-2-hydroxy-6-oxo-piperidin-1-ylmethyl)-3-oxo -3-(2-pyridin-4-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-propane base]-tert-butyl carbamate

Similar to the procedure of Preparation Example 6, (3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-yl synthesized in Preparation Example 4 )-butyric acid (24mg, 0.07mmol) and 2-pyridin-4-yl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[ 3,4-d] Pyrimidine hydrochloride (30 mg) was coupled to obtain 13.5 mg (yield: 32%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.37(m, 9H), 2.16-2.26(m, 2H), 2.59-2.88(m, 5H), 3.01-3.22(m, 3H), 3.37-3.94 (m, 3H), 4.30(m, 1H), 4.92-5.01(m, 3H), 8.28(m, 2H), 8.75(m, 1H); MS(m/e) 637(M+Na)

Example 6: Synthesis of 1-[(S)-2-amino-4-oxo-4-(2-pyridin-4-yl-4-trifluoromethyl-5,8- Dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one salt salt

The compound synthesized in Preparation Example 9 was treated with commercially available dioxane containing 4.0N hydrogen chloride and stirred for 1 hour. Since the title compound was a diastereomeric compound, the reaction solution was distilled under reduced pressure and separated into two isomers by prep-TLC. In addition to the isolated isomer, 2 mg of the relatively less polar title compound was obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ2.17(m, 1H), 2.58-2.68(m, 4H), 2.77-2.82(m, 1H), 3.11-3.21(m, 2H), 3.34-3.70 (m, 2H), 3.91-4.00(m, 3H), 4.77-5.06(m, 3H), 8.44(d, 2H, J=6.0MHz), 8.76(d, 2H, J=4.8MHz); MS( m/e)515(M+1)

Example 7: Synthesis of 1-[(S)-2-amino-4-oxo-4-(2-pyridin-4-yl-4-trifluoromethyl-5,8- Dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one salt salt

The compound synthesized in Preparation Example 9 was treated with commercially available dioxane containing 4.0N hydrogen chloride and stirred for 1 hour. Since the title compound was a diastereomeric compound, the reaction solution was distilled under reduced pressure and separated into two isomers by prep-TLC. In addition to the isolated isomer, 1.8 mg of the relatively polar title compound were obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ2.17(m, 1H), 2.53-2.65(m, 4H), 2.74-2.80(m, 1H), 3.06-3.20(m, 2H), 3.34-3.66 (m, 2H), 3.82-4.00(m, 3H), 4.77-5.00(m, 3H), 8.43(d, 2H, J=6.0MHz), 8.76(d, 2H, J=4.4MHz); MS( m/e)515(M+1)

Preparation Example 10: Synthesis of [(S)-1-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-ylmethyl)-3-(2- Furan-2-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-3-oxo-propane base]-tert-butyl carbamate

Similar to the procedure of Preparation Example 6, (3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-yl synthesized in Preparation Example 4 )-butyric acid (25mg, 0.07mmol) and 2-furan-2-yl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[ 3,4-d] Pyrimidine hydrochloride (30 mg) was coupled to obtain 18.1 mg (yield: 43%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.28(m, 9H), 2.13(m, 1H), 2.39-2.47(m, 3H), 2.61-2.76(m, 2H), 2.89-3.03(m , 3H), 3.56-3.80(m, 3H), 4.31(m, 1H), 4.70-4.86(m, 3H), 6.55-6.56(m, 1H), 7.28(d, 1H, J=4.0MHz), 7.52-7.57 (m, 1H); MS (m/e) 626 (M+Na)

Example 8: Synthesis of 1-[(S)-2-amino-4-(2-furan-2-yl-4-trifluoromethyl-5,8-dihydro -6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-4-oxo-butyl]-5,5-difluoro-6-hydroxyl-piperidine-2- Ketone hydrochloride

Similar to the procedure of Example 6, two isomers were separated from the compound synthesized in Preparation Example 10. In addition to the isolated isomers, 4.7 mg of the relatively less polar title compound were obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ2.17(m, 1H), 2.45-2.81(m, 5H), 3.04-3.17(m, 2H), 3.33-3.72(m, 2H), 3.87-3.96 (m, 3H), 4.90-5.00(m, 3H), 6.68(s, 1H), 7.41(d, 1H, J=3.2MHz), 7.79(s, 1H); MS(m/e) 504(M +1)

Example 9: Synthesis of 1-[(S)-2-amino-4-(2-furan-2-yl-4-trifluoromethyl-5,8-dihydro -6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-4-oxo-butyl]-5,5-difluoro-6-hydroxyl-piperidine-2- Ketone hydrochloride

Similar to the procedure of Example 6, two isomers were separated from the compound synthesized in Preparation Example 10. In addition to the isolated isomer, 4.8 mg of the relatively polar title compound were obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ2.17(m, 1H), 2.51-2.76(m, 5H), 3.03-3.11(m, 2H), 3.34-3.70(m, 2H), 3.84-3.93 (m, 3H), 4.82-4.90(m, 3H), 6.68(s, 1H), 7.41(d, 1H, J=3.6MHz), 7.79(s, 1H); MS(m/e) 504(M +1)

Preparation Example 11: Synthesis of {(S)-1-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-ylmethyl Base)-3-[2-(2-methoxy-ethyl)-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine -7-yl]-3-oxo-propyl}-tert-butyl carbamate

Similar to the procedure of Preparation Example 6, (3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-yl synthesized in Preparation Example 4 )-butyric acid (61mg, 0.17mmol) and 2-(2-methoxy-ethyl)-4-trifluoromethyl-5,6,7,8-tetrafluoromethyl synthesized in Preparation 2-(6) Hydrogen-pyrido[3,4-d]pyrimidine hydrochloride (65 mg) was coupled to obtain 38.2 mg (yield: 37%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.31-1.44 (m, 9H), 2.17-2.24 (m, 2H), 2.58-3.74 (m, 4H), 2.88-3.37 (m, 10H), 3.85 -3.98(m, 4H), 4.29(m, 1H), 4.75-4.96(m, 3H); MS(m/e) 618(M+Na)

Example 10: Synthesis of 1-{(S)-2-amino-4-[2-(2-methoxy-ethyl)-4-trifluoromethyl -5,8-Dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl]-4-oxo-butyl}-5,5-difluoro-6-hydroxy-piper Pyridine-2-one hydrochloride

Similar to the procedure of Example 6, two isomers were separated from the compound synthesized in Preparation Example 11. In addition to the isolated isomer, 6.2 mg of the title compound was obtained as relatively less polar.

¹ H NMR (400MHz, MeOH-d ₄ ) δ2.18(m, 1H), 2.62-2.78(m, 4H), 3.02-3.42(m, 10H), 3.68-4.06(m, 5H), 4.80-4.90 (m,3H); MS(m/e)496(M+1)

Example 11: Synthesis of 1-{(S)-2-amino-4-[2-(2-methoxy-ethyl)-4-trifluoromethyl -5,8-Dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl]-4-oxo-butyl}-5,5-difluoro-6-hydroxy-piper Pyridine-2-one hydrochloride

Similar to the procedure of Example 6, two isomers were separated from the compound synthesized in Preparation Example 11. In addition to the isolated isomer, 9.1 mg of the relatively polar title compound were obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ2.21(m, 1H), 2.60-2.64(m, 3H), 2.87-3.37(m, 11H), 3.61-4.02(m, 5H), 4.79-4.99 (m,3H); MS(m/e)496(M+1)

Preparation 12: Synthesis of {(S)-1-(3,3-difluoro-2-hydroxy-6-oxo-piperidin-1-ylmethyl)-3- Oxygen-3-(2-thiophene)-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)- Propyl]-tert-butyl carbamate

Similar to the procedure of Preparation Example 6, (3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-yl synthesized in Preparation Example 4 )-butyric acid (39mg, 0.11mmol) and 2-thien-3-yl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[ 3,4-d] Pyrimidine hydrochloride (50 mg) was coupled to obtain 26 mg (yield: 38%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.40(s, 9H), 2.16-2.25(m, 2H), 2.59-2.75(m, 5H), 3.01-3.13(m, 3H), 3.83-3.93 (m, 3H), 4.31(m, 1H), 4.88-4.98(m, 3H), 7.51-7.53(m, 1H), 7.89(d, 1H, J=4.4MHz), 8.40(s, 1H); MS(m/e)642(M+Na)

Example 12: Synthesis of 1-[(S)-2-amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8- two Hydrogen-6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one salt salt

Similar to the procedure of Example 6, two isomers were separated from the compound synthesized in Preparation Example 12. In addition to the isolated isomer, 6.2 mg of the title compound was obtained as relatively less polar.

¹ H NMR (400MHz, MeOH-d ₄ ) δ2.18(m, 1H), 2.58-2.67(m, 4H), 2.77-2.81(m, 1H), 3.04-3.12(m, 2H), 3.34-3.42 (m, 2H), 3.81-3.96(m, 3H), 4.90-4.97(m, 3H), 7.52-7.55(m, 1H), 7.89(d, 1H, J=5.2MHz), 8.40-8.41(m , 1H); MS(m/e)520(M+1)

Example 13: Synthesis of 1-[(S)-2-amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8- Dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one salt salt

Similar to the procedure of Example 6, two isomers were separated from the compound synthesized in Preparation Example 12. In addition to the isolated isomer, 5.8 mg of the relatively polar title compound were obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ2.17(m, 1H), 2.55-2.65(m, 4H), 2.80-2.85(m, 1H), 3.04-3.12(m, 2H), 3.46-3.51 (m, 1H), 3.66-3.74(m, 1H), 3.86-3.95(m, 3H), 4.85-4.93(m, 3H), 7.52-7.55(m, 1H), 7.89(d, 1H, J= 5.2MHz), 8.40-8.41(m, 1H); MS(m/e)520(M+1)

Preparation Example 13: Synthesis of {(S)-1-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-ylmethyl Base)-3-[2-(4-fluoro-phenyl)-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine-7- base]-3-oxo-propyl}-tert-butyl carbamate

Similar to the procedure of Preparation Example 5, (3S)-tert-butoxycarbonylamino-4-(3,3-difluoro-2-hydroxyl-6-oxo-piperidin-1-yl synthesized in Preparation Example 4 )-butyric acid (30mg, 0.09mmol) and 2-(4-fluoro-phenyl)-4-trifluoromethyl-5,6,7,8-tetrahydro- Pyrido[3,4-d]pyrimidine hydrochloride (25 mg) was coupled to obtain 10.5 mg (yield: 20%) of the title compound.

¹ H NMR (500MHz, CDCl ₃ ) δ1.41-1.44 (m, 9H), 2.57-2.61 (m, 6H), 3.12-3.14 (m, 3H), 3.91-4.00 (m, 4H), 4.86-4.96 (m, 4H), 7.16-7.18(m, 2H), 8.46-8.49(m, 2H); MS(m/e) 654(M+Na)

Example 14: Synthesis of 1-{(S)-2-amino-4-[2-(4-fluoro-phenyl)-4-trifluoromethyl-5,8- Dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl]-4-oxo-butyl}-5,5-difluoro-6-hydroxy-piperidine-2- Ketone hydrochloride

Using the compound synthesized in Preparation Example 13 similarly to the procedure of Example 3, 8.8 mg (yield: 93%) of the title compound were obtained.

¹ H NMR (500MHz, MeOH-d ₄ ) δ2.55-2.64(m, 6H), 3.00-3.38(m, 5H), 3.86-3.93(m, 2H), 4.84-4.92(m, 3H), 7.21 -7.24(m, 2H), 8.48-8.51(m, 2H); MS(m/e) 532(M+1)

Preparation 14: Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3,3-difluoro-2,6-dioxo-piperene Pyridin-1-yl)-butanoic acid

(1) Synthesis of (S)-3-tert-butoxycarbonylamino-4-hydroxy-butyric acid benzyl ester

(S)-2-tert-butoxycarbonylamino-succinic acid 4-benzyl ester (545 mg, 1.69 mmol) was dissolved in tetrahydrofuran (THF), to which was added N-methylmorpholine ( NMM, 0.19 mL, 1.77 mmol) and isobutyl chloroformate (0.23 mL, 1.77 mmol), then stirred at the temperature for 30 minutes. After 30 minutes, the resulting white solid salt was removed by filtration through celite, and a solution of sodium borohydride (96 mg, 2.53 mmol) in distilled water (2 mL) was added to the resulting clear filtrate at room temperature without further concentration . After stirring at room temperature for 1 hour, THF was distilled off under reduced pressure. The reaction was diluted with ethyl acetate and quenched by adding 1N HCl. The organic layer was extracted with ethyl acetate and purified by column chromatography (ethyl acetate:hexane=1:1), thereby obtaining 468 mg (yield: 90%) of the title compound.

¹ H NMR (500MHz, CDCl ₃ ) δ1.42(s, 9H), 2.67(d, 2H, J=5.5Hz), 3.67-3.68(m, 2H), 3.99-4.01(m, 1H), 5.12( s, 2H), 5.29 (b, 1H), 7.33-7.36 (m, 5H); MS (m/e) 332 (M+Na)

(2) Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3,3-difluoro-2,6-dioxo-piperidine-1- base)-benzyl butyrate

Similar to the procedure of Preparation 4-(2), use (S)-3-tert-butoxycarbonylamino-4-hydroxy-butyric acid benzyl ester (1.5 g, 4.9 mmol) synthesized in Section (1) and 3,3-difluoropiperidine-2,6-dione (880 mg, 5.9 mmol) synthesized in Preparation Example 3, to obtain 1.86 g (yield: 86%) of the title compound.

¹ H NMR (500MHz, CDCl ₃ ) δ1.37(s, 9H), 2.43(m, 2H), 2.62-2.67(m, 2H), 2.88-2.89(m, 2H), 3.71(d, 1H, J =11Hz), 4.2(m, 2H), 5.17(s, 2H), 5.19(d, 1H), 7.36-7.37(m, 5H)

(3) Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3,3-difluoro-2,6-dioxo-piperidine-1- base)-butyric acid

(S)-3-tert-butoxycarbonylamino-4-(3,3-difluoro-2,6-dioxo-piperidin-1-yl)-butyric acid benzyl The base ester (310 mg, 0.70 mmol) was dissolved in methanol and 10% Pd/C (31 mg, 0.1 equiv) was added thereto. The mixture was stirred under 1 atmosphere of hydrogen for 1 hour. After completion of the reaction, the solid matter was removed by celite filtration to obtain 260 mg of the crude title compound.

Preparation 15: Synthesis of [(S)-3-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d] Pyrimidin-7-yl)-1-(3,3-difluoro-2,6-dioxo-piperidin-1-ylmethyl)-3-oxo-propyl]-carbamic acid tert-butyl ester

Similar to the procedure of Preparation Example 5, using (S)-3-tert-butoxycarbonylamino-4-(3,3-difluoro-2,6-dioxo-piperidine-1- base)-butyric acid (30mg, 0.09mmol) and 2,4-bis-trifluoromethyl-5,6,7,8- tetrahydropyrido [3,4-d]pyrimidine salt synthesized in Preparation Example 1 acid salt (23 mg, 0.009 mmol) to obtain 6.6 mg (yield: 13%) of the title compound.

¹ H NMR (500MHz, CDCl ₃ ) δ1.37(s, 9H), 2.42(m, 2H), 2.76-2.95(m, 4H), 3.14-3.28(m, 2H), 3.71-4.22(m, 5H) ), 4.85-5.00(m, 2H), 5.52-5.55(m, 1H); MS(m/e)626(M+1)

Example 15: Synthesis of 1-[(S)-2-amino-4-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyridine Pyrido[3,4-d]pyrimidin-7-yl)-4-oxo-butyl]-3,3-difluoro-piperidine-2,6-dione

Using the compound synthesized in Preparation Example 15 similarly to the procedure of Example 3, 6.4 mg (yield: 97%) of the title compound was obtained.

¹ H NMR (500MHz, MeOH-d ₄ ) δ2.51-2.54(m, 2H), 2.85-2.91(m, 4H), 3.12-3.21(m, 3H), 4.10-4.20(m, 4H), 4.95 (m,2H); MS(m/e)504(M+1)

Preparation 16: Synthesis of 3-fluoro-piperidine-2,6-dione

(1) Synthesis of 2-fluoro-diethyl glutarate

Similar to the procedure of Preparation 3-(1), commercially available ethyl bromofluoroacetate (7.1 mL, 60 mmol) and ethyl acrylate (3.3 mL, 30 mmol) were used to obtain 1.86 g (yield: 30%) the title compound.

¹ H NMR (400MHz, CDCl ₃ ) δ1.25-1.37(m, 6H), 2.17-2.37(m, 2H), 2.51-2.57(m, 2H), 4.14-4.33(m, 4H), 4.93-5.08 (m, 1H)

(2) Synthesis of 4-carbamoyl-4-fluoro-butyric acid ethyl ester

Using the 2-fluoro-glutaric acid diethyl ester (2.4 g, 11.7 mmol) synthesized in section (1) similarly to the procedure of Preparation Example 3-(2), 2.2 g of the crude title compound were obtained.

¹ H NMR (400MHz, CDCl ₃ ) δ1.21-1.33(m, 3H), 2.13-2.38(m, 2H), 2.47-2.52(m, 2H), 4.13-4.18(m, 2H), 4.89-5.08 (m, 1H), 5.90-6.32(b, 1H)

(3) Synthesis of 3-fluoro-piperidine-2,6-dione

Similar to the procedure of Preparation 3-(3), 720 mg was obtained using 4-carbamoyl-4-fluoro-butyric acid ethyl ester (2.2 g, 12.5 mmol) synthesized in section (2) (yield: 44%) the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ2.18-2.41 (m, 2H), 2.71-2.75 (m, 2H), 5.11-5.27 (m, 1H); MS (m/e) 132 (M+ 1)

Preparation 17: Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2-hydroxy-6-oxo-piperidine -1-yl)-butanoic acid

(1) Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2,6-dioxo-piperidin-1-yl)-butanol isopropyl ester

Similar to the procedure of Preparation 4-(2), 3-fluoro-piperidine-2,6-dione (47 mg, 0.36 mmol) synthesized in Preparation 16 and ( 3S)-Isopropyl tert-butoxycarbonylamino-4-hydroxybutyrate (78.4 mg, 0.3 mmol) afforded 139 mg of crude title compound.

¹ H NMR (400MHz, CDCl ₃ ) δ1.24-1.39(m, 6H), 1.44(s, 9H), 2.24-2.35(m, 2H), 2.48-2.71(m, 2H), 2.85-2.91(m , 2H), 3.73(d, 1H, J=4.8Hz), 4.09-4.20(m, 2H), 4.99-5.31(m, 3H); MS(m/e) 397(M+Na)

(2) Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2-hydroxyl-6-oxo-piperidin-1-yl)- Isopropyl Butyrate

Similar to the procedure of Preparation 4-(3), using (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2,6-dioxo-piperidine synthesized in Section (1) -1-yl)-isopropyl butyrate (139mg, 0.37mmol) to obtain 109mg (yield: 78%) of the title compound.

MS(m/e)399(M+Na)

(3) Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2-hydroxyl-6-oxo-piperidin-1-yl)- butyric acid

Similar to the procedure of Preparation 4-(4), using (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2-hydroxyl-6-oxo-piperene synthesized in Section (2) Pyridin-1-yl)-isopropyl butyrate (102mg, 0.27mmol) gave 54mg (yield: 60%) of the title compound.

MS(m/e)357(M+Na)

Preparation Example 18: Synthesis of [(S)-3-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido [3,4-d]pyrimidin-7-yl)-1-(3-fluoro-2-hydroxyl-6-oxo-piperidin-1-ylmethyl)-3-oxo-propyl]-amino tert-butyl formate

Similar to the procedure of Preparation Example 6, using (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2-hydroxyl-6-oxo-piperidin-1-yl) synthesized in Preparation Example 17 -Butyric acid (38mg, 0.11mmol) and 2-tert-butyl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3,4 synthesized in Preparation Example 2-(2) -d] Pyrimidine hydrochloride (49 mg, 0.16 mmol), to obtain 42 mg (yield: 65%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.41(d, 18H), 2.17(m, 2H), 2.45(m, 2H), 2.71(m, 2H), 3.00(m, 1H), 3.11( m, 2H), 3.92(m, 3H), 4.11(m, 1H), 4.88-4.91(m, 3H) 5.43-5.55(m, 1H); MS(m/e) 598(M+Na)

Example 16: Synthesis of 1-[(S)-2-amino-4-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro -6H-pyrido[3,4-d]pyrimidin-7-yl)-4-oxo-butyl]-5-fluoro-6-hydroxy-piperidin-2-one hydrochloride

Using the compound synthesized in Preparation Example 18 similarly to the procedure of Example 3, 6.0 mg (yield: 18%) of the title compound was obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.42(s, 9H), 2.00-2.50(m, 4H), 2.81-3.11(m, 5H), 3.50-3.93(m, 4H), 4.84-5.10 (m,3H)5.48-5.62(m,1H); MS(m/e)476(M+1)

Preparation Example 19: Synthesis of [(S)-1-(3-fluoro-2-hydroxy-6-oxo-piperidin-1-ylmethyl)-3-(2-fur Pyran-2-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-3-oxo-propyl]- tert-butyl carbamate

Similar to the procedure of Preparation Example 6, using (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2-hydroxyl-6-oxo-piperidin-1-yl) synthesized in Preparation Example 17 -Butyric acid (35mg, 0.11mmol) and 2-furan-2-base-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3 synthesized in Preparation Example 2-(5) , 4-d] pyrimidine hydrochloride (45mg, 0.15mmol), obtained 49mg (yield: 79%) of the title compound

MS(m/e)608(M+Na)

Example 17: Synthesis of 1-[(S)-2-amino-4-(2-furan-2-yl-4-trifluoromethyl-5,8-di Hydrogen-6H-pyrido[3,4-d]pyrimidin-7-yl)-4-oxo-butyl]-5-fluoro-6-hydroxy-piperidin-2-one hydrochloride Salt

Similar to the procedure of Example 3, using the compound synthesized in Preparation Example 19, 15 mg (yield: 35%) of the title compound was obtained.

¹ H NMR (400 MHz, MeOH-d ₄ ) δ2.17-3.14 (m, 9H), 3.70-3.89 (m, 4H), 4.86-4.87 (m, 3H), 5.43-5.55 (m, 1H), 6.68 ( m, 1H), 7.41(m, 1H), 7.79(m, 1H); MS(m/e) 486(M+1)

Preparation 20: Synthesis of [(S)-1-(3-fluoro-2-hydroxy-6-oxo-piperidin-1-ylmethyl)-3-oxo -3-(2-thiophen-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-propane base]-tert-butyl carbamate

Similar to the procedure of Preparation Example 6, using (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2-hydroxyl-6-oxo-piperidin-1-yl) synthesized in Preparation Example 17 -Butyric acid (48mg, 0.14mmol) and 2-thien-3-yl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3 synthesized in Preparation Example 2-(7) , 4-d] pyrimidine hydrochloride (65mg, 0.20mmol), to obtain 13mg (yield: 15%) of the title compound.

MS(m/e)624(M+Na)

Example 18: Synthesis of 1-[(S)-2-amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8- Dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-5-fluoro-6-hydroxy-piperidin-2-one hydrochloride

Using the compound synthesized in Preparation Example 20 similarly to the procedure of Example 3, 1.4 mg (yield: 12%) of the title compound was obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ2.2-2.3(m, 4H), 2.50(m, 1H), 3.00-3.15(m, 4H), 3.74(m, 1H), 3.88(m, 3H ), 4.88(m, 3H), 5.51(m, 1H), 7.53(m, 1H), 7.88(m, 1H), 8.39(m, 1H); MS(m/e) 502(M+1)

Preparation 21: Synthesis of [(S)-1-(3-fluoro-2-hydroxy-6-oxo-piperidin-1-ylmethyl)-3-oxo -3-(2-Propyl-5-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-propyl]-ammonia tert-butyl carbamate

Similar to the procedure of Preparation Example 6, (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2-hydroxyl-6-oxo-piperidin-1-yl synthesized in Preparation Example 17 was used )-butyric acid (47mg, 0.14mmol) and 2-propyl-4-trifluoromethyl-5,6,7,8-tetrahydropyrido[3,4- d] Pyrimidine hydrochloride (56 mg, 0.20 mmol), to obtain 15 mg (yield: 18%) of the title compound.

MS(m/e)584(M+Na)

Example 19: Synthesis of 1-[(S)-2-amino-4-oxo-4-(2-propyl-5-trifluoromethyl-5,8-di Hydrogen-6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-5-fluoro-6-hydroxy-piperidin-2-one hydrochloride

Using the compound synthesized in Preparation Example 21 similarly to the procedure of Example 3, 2.3 mg (yield: 18%) of the title compound was obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ0.95-0.99(m, 3H), 1.82-1.84(m, 2H), 2.18(m, 2H), 2.44-2.56(m, 3H), 2.74(m , 1H), 2.90-3.07(m, 5H), 3.76-3.89(m, 4H), 4.84(m, 3H), 5.54(m, 1H); MS(m/e) 462(M+1)

Preparation 22: Synthesis of [(S)-1-(3-fluoro-2-hydroxy-6-oxo-piperidin-1-ylmethyl)-3-oxo -3-(2-pyridin-4-yl-5-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-propane base]-tert-butyl carbamate

Similar to the procedure of Preparation Example 6, using (S)-3-tert-butoxycarbonylamino-4-(3-fluoro-2-hydroxyl-6-oxo-piperidin-1-yl) synthesized in Preparation Example 17 -Butyric acid (33mg, 0.10mmol) and 2-pyridin-4-yl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3 synthesized in Preparation Example 2-(4) , 4-d] pyrimidine hydrochloride (44 mg, 0.14 mmol), to obtain 55 mg (yield: 65%) of the title compound.

MS(m/e)619(M+Na)

Example 20: Synthesis of 1-[(S)-2-amino-4-oxo-4-(2-pyridin-4-yl-5-trifluoromethyl-5,8- Dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-5-fluoro-6-hydroxy-piperidin-2-one hydrochloride

Using the compound synthesized in Preparation Example 22 similarly to the procedure of Example 3, 12 mg (yield: 24%) of the title compound was obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.30-3.21(m, 8H), 3.37-4.20(m, 5H), 5.00(m, 3H), 8.43(s, 2H), 8.76(s, 2H ); MS (m/e) 519 (M+Na)

Preparation 23: Synthesis of 3-methyl-piperidine-2,6-dione

To commercially available 2-methylglutaric acid (1 g, 6.8 mmol) was added acetic anhydride at room temperature, and the mixture was stirred at 60° C. under reflux for 8 hours. After the completion of the reaction was confirmed by TLC, the remaining acetic anhydride was removed under reduced pressure. The concentrated compound was dissolved in tetrahydrofuran and ammonia aqueous solution (1.7 mL, 14.6 mmol) was slowly added thereto at 0°C, followed by stirring at room temperature for 8 hours. After the reaction was completed, the remaining aqueous ammonia solution was removed under reduced pressure and acetic anhydride was added, followed by reflux at 60° C. for 8 hours. Residual acetic anhydride was removed under reduced pressure. The concentrate was purified by column chromatography (ethyl acetate:hexane=1:1) to obtain 682 mg (yield: 78%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.24-1.28(m, 3H), 1.71-1.77(1H), 2.04-2.08(m, 1H), 2.57-2.65(m 3H); MS(m/ e)128(M+1)

Preparation 24: Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3-methyl-2,6-dioxo-piperidine -1-yl)-butanoic acid

(1) Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3-methyl-2,6-dioxo-piperidin-1-yl)- Benzyl butyrate

Similar to the procedure of Preparation 4-(2), 3-methyl-piperidine -2,6-dione (53 mg, 0.42 mmol) synthesized in Preparation 23 and 3-methyl-piperidine synthesized in Preparation 14-(1) were used. (S)-3-tert-butoxycarbonylamino -4-hydroxy-butyric acid benzyl ester (107 mg, 0.35 mmol) to obtain 129 mg (yield: 88%) of the title compound.

¹ H NMR (400MHz, CDCl ₃ ) δ1.30-1.35(m, 3H), 1.47(s, 9H), 1.78-2.01(m, 2H), 2.53-2.85(m, 5H), 3.72(m, 1H) ), 4.13-4.36(m, 2H), 5.12-5.23(m, 3H), 7.30-7.39(m, 5H)

MS(m/e)441(M+Na)

(2) Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3-methyl-2,6-dioxo-piperidin-1-yl)- butyric acid

(S)-3-tert-butoxycarbonylamino-4-(3-methyl-2,6-dioxo- piperidin-1-yl)-butyric acid benzyl ester synthesized in section (1) (218mg, 0.52mmol) was dissolved in methanol and 10% Pd/C (22mg, 0.1eq) was added thereto, followed by stirring under 1 atm of hydrogen for 1 hour. After the reaction was complete, the Pd on the charcoal was removed by celite filtration. The residue was purified by prep-TLC to obtain 59 mg (yield: 35%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.28(m, 3H), 1.42(s, 9H), 1.80-1.99(m, 2H), 2.38-2.87(m, 5H), 3.72(m, 1H ), 4.22-4.44 (m, 2H); MS (m/e) 351 (M+Na)

Preparation 25: Synthesis of [(S)-1-(3-methyl-2,6-dioxo-piperidin-1-ylmethyl)-3-oxo-3-(2- Thiophen-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-propyl]-ammonia tert-butyl carbamate

Similar to the procedure of Preparation Example 6, using (S)-3-tert-butoxycarbonylamino-4-(3-methyl-2,6-dioxo-piperidin-1-yl) synthesized in Preparation Example 24 -butyric acid (29mg, 0.09mmol) and 2-thien-3-yl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3 synthesized in Preparation Example 2-(7) , 4-d] pyrimidine hydrochloride (40mg, 0.12mmol), to obtain 47mg (yield: 88%) of the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.24-1.28(m, 3H), 1.38(s, 9H), 1.68-2.04(m, 2H), 2.69-2.94(m, 5H), 3.02-3.17 (m, 2H), 3.91(m, 2H), 3.72(m, 1H), 4.11-4.20(m, 3H), 4.80-4.90(m, 2H), 7.53(m, 1H), 7.91(m, 1H ), 8.41 (m, 1H); MS (m/e) 618 (M+Na)

Example 21: Synthesis of 1-[(S)-2-amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8- Dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-butyl]-3-methyl-piperidine-2,6-dione hydrochloride

Using the compound synthesized in Preparation Example 25 similarly to the procedure of Example 3, 23 mg (yield: 65%) of the title compound were obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.23-1.31(m, 3H), 1.72-2.17(m, 2H), 2.64-2.72(m, 5H), 3.04-3.13(m, 2H), 3.55 -3.68(m, 2H), 3.87-3.93(m, 2H), 4.17(m, 1H), 4.90(m, 2H), 7.53(m, 1H), 7.90(m, 1H), 8.41(m, 1H ); MS(m/e)496(M+1)

Preparation 26: Synthesis of 3-methyl-pyrrolidine-2,5-dione

(1) Synthesis of 2-methyl-succinic acid

Commercially available dimethyl methylsuccinate (1.57g, 9.8mmol) was dissolved in a 3:1 mixed solvent of tetrahydrofuran and water, lithium hydroxide (4g, 98mmol) was added and stirred at 40°C under reflux 2 days. After the completion of the reaction was confirmed by TLC, the residue was distilled under reduced pressure to obtain the title compound.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.21-1.23 (d, 3H, J=7.2Hz), 2.43-2.48 (m, 1H), 2.64-2.70 (m, 1H), 2.80-2.89 (m , 1H)

(2) Synthesis of 3-methyl-pyrrolidine-2,5-dione

Similar to the procedure of Preparation 23, using 2-methyl-succinic acid synthesized in section (1), 250 mg of the title compound were obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.19-1.29 (m, 3H), 2.32-2.40 (m, 1H), 2.71-2.96 (m, 2H)

Preparation 27: Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3-methyl-2,5-dioxo-pyrrole Alk-1-yl)-butyric acid

(1) Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3-methyl-2,5-dioxo-pyrrolidine-1- base)-benzyl butyrate

Similar to the procedure of Preparation 4-(2), use 3-methyl-pyrrolidine-2,5-dione (61 mg, 0.54 mmol) synthesized in Preparation 26 and 3-methyl-pyrrolidine synthesized in Preparation 14-(1) (S)-Benzyl 3-tert-butoxycarbonylamino-4-hydroxy-butyrate (140 mg, 0.45 mmol) afforded 230 mg of crude title compound.

¹ H NMR (400MHz, CDCl ₃ ) δ1.31-1.39(m, 3H), 1.43(s, 9H), 2.29-2.39(m, 1H), 2.62-2.92(m, 4H), 3.70(m, 1H) ), 4.18(m, 2H), 5.13-5.14(m, 2H), 5.24(b, 1H), 7.35-7.36(m, 5H); MS(m/e) 427(M+Na)

(2) Synthesis of (S)-3-tert-butoxycarbonylamino-4-(3-methyl-2,5-dioxo-pyrrolidine-1- base)-butyric acid

Similar to the procedure of Preparation 23-(2), using (S)-3-tert-butoxycarbonylamino-4-(3-methyl-2,5-dioxo-pyrrole synthesized in Section (1) Alk-1-yl)-benzyl butyrate (218 mg, 0.52 mmol) afforded 62 mg of the title compound.

MS(m/e)337(M+Na)

Preparation 28: Synthesis of [(S)-3-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido [3,4-d]pyrimidin-7-yl)-1-(3-methyl-2,5-dioxo-pyrrolidin-1-ylmethyl)-3-oxo-propyl]-ammonia tert-butyl carbamate

Similar to the procedure of Preparation Example 6, using (S)-3-tert-butoxycarbonylamino-4-(3-methyl-2,5-dioxo-pyrrolidin-1-yl) synthesized in Preparation Example 27 -Butyric acid (39mg, 0.13mmol) and 2-tert-butyl-4-trifluoromethyl-5,6,7,8-tetrahydro-pyrido[3,4 synthesized in Preparation 2-(2) -d] Pyrimidine hydrochloride (52 mg, 0.18 mmol), 74 mg of the title compound was quantitatively obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.24-1.28(m, 3H), 1.38-1.42(m, 18H), 2.27-2.92(m, 5H), 3.00-3.12(m, 2H), 3.63 -4.59(m, 5H), 4.82-4.86(m, 2H); MS(m/e) 578(M+Na)

Example 22: Synthesis of 1-[(S)-2-amino-4-(2- tert -butyl-4-trifluoromethyl-5,8-dihydro -6H-pyrido[3,4-d] Pyrimidin-7-yl)-4-oxo-butyl]-3-methyl-pyrrolidine-2,6-dione hydrochloride

Using the compound synthesized in Preparation Example 28 similarly to the procedure of Example 3, 32 mg (yield: 61%) of the title compound were obtained.

¹ H NMR (400MHz, MeOH-d ₄ ) δ1.26-1.29(m, 3H), 1.42(s, 9H), 2.33-2.38(m, 1H), 2.57-2.67(m, 2H), 2.90-2.92 (m, 2H), 3.00-3.10(m, 2H), 3.55-3.37(m, 3H), 3.84-3.90(m, 2H), 4.82-4.86(m, 2H); MS(m/e) 456( M+1)

Experimental example 1: Analysis of the inhibitory activity of DPP-IV

Dipeptidyl peptidase-IV (DPP -IV), said improved method comprising cloning, purification using baculovirus, and activation steps. Candidate inhibitors were tested for potency using DPP-IV as described below. Cloned DPP-IV was expressed in baculovirus, purified by nickel column and then dialyzed. The inhibitors synthesized in the examples were tested using the fluorescent substrate Ac-Gly-Pro-AFC to determine their DPP-IV inhibitory activity. Various concentrations of inhibitors were subjected to enzymatic reactions using 100 μM Ac-Gly-Pro-AFC in a buffer solution containing 50 mM HEPES (pH 7.4) at 25° C., and the concentration of DPP-IV was 7.1 nM. After 1 hour of enzyme reaction, the _IC50 value of the inhibitor was determined by measuring the amount of emitted fluorescence in a spectrofluorometer, and then calculating the inhibitor concentration showing 50% inhibition of the total enzyme reaction. A SpectraMAX GeminiXS spectrofluorometer (Molecular Device Co.) was used as a spectrofluorometer, and excitation and emission wavelengths were set to 400 nm and 505 nm, respectively. The results obtained are summarized in Table 1 below.

Table 1

IC ₅₀ (nM) IC ₅₀ (nM) Example 1 36 Example 2 5 Example 3 20 Example 4 39 Example 5 17 Example 6 10 Example 7 63 Example 8 6 Example 9 93 Example 10 14 Example 11 160 Example 12 10 Example 13 195 Example 14 57 Example 15 2500 Example 16 197 Example 17 81 Example 18 159 Example 19 409 Example 20 206 Example 21 ＞2500 Example 22 ＞2500

Experimental example 2: Analysis of the inhibitory activity of DPP-II, VIII and IX

In order to compare the DPPase selectivity of certain compounds of Examples 1 to 22 with known DPP-IV inhibitors, the enzyme inhibitory activity of these test compounds was measured as follows.

- Measurement of DPP-II inhibitory activity: DPP-II was obtained and purified. The DPP-II inhibitory activity of the DPP-IV inhibitors listed in Table 2 below was analyzed using Gly-Pro-AFC as a substrate. Various concentrations of DPP-IV inhibitors were subjected to enzyme reactions at room temperature using 0.018 µg DPP-II and 100 µM Gly-Pro-AFC in a buffer solution (pH 4.5) containing 50 mM acetic acid. _IC50 values for inhibitors were determined by following the reaction rate for 60 minutes using a spectrofluorometer and analyzing the results using TableCurve 2D (v 5.01). DPP-IV inhibitors were analyzed using a spectrofluorometer at excitation and emission wavelengths of 405nm and 505nm, respectively.

- Measurement of DPP-VIII inhibitory activity: DPP-VIII is obtained and purified. The DPP-IV inhibitors listed in Table 2 below were analyzed for their DPP-VIII inhibitory activity using Gly-Pro-AFC as a substrate. Enzyme reactions were performed on various concentrations of DPP-IV inhibitors using 0.0049 μg DPP-VIII and 100 μM Gly-Pro-AFC in a buffer solution (pH 7.5) containing 50 mM HEPES at room temperature. _IC50 values for inhibitors were determined by following the reaction rate for 60 minutes using a spectrofluorometer and analyzing the results using TableCurve 2D (v 5.01). DPP-IV inhibitors were analyzed using a spectrofluorometer at excitation and emission wavelengths of 405nm and 505nm, respectively.

- Measurement of DPP-IX inhibitory activity: DPP-IX was obtained and purified. The DPP-IV inhibitors listed in Table 2 below were analyzed for their DPP-IX inhibitory activity using Gly-Pro-AFC as a substrate. Various concentrations of DPP-IV inhibitors were subjected to enzyme reactions at room temperature using 200 ng DPP-IX and 100 μM Gly-Pro-AFC in a buffer solution (pH 7.5) containing 50 mM HEPES. The _IC50 values of the inhibitors were determined by tracking the reaction rate in real time using a spectrofluorometer and analyzing the results using TableCurve 2D (v 5.01). DPP-IV inhibitors were analyzed using a spectrofluorometer at excitation and emission wavelengths of 405nm and 505nm, respectively.

The analytical results obtained are summarized in Table 2 below.

Table 2

Inhibitor DPP-II (μM) DPP-IV(nM) DPP-VIII(μM) DPP-IX (μM) Example 2 ＞400 5 ＞400 ＞400 Example 5 ＞161 17 ＞400 ＞400 Example 6 ＞70 10 ＞127 ＞100 Example 8 ＞80 6 ＞400 ＞130 Example 10 ＞400 14 ＞400 ＞210 Example 12 ＞50 10 ＞400 ＞110 Example ^83a ＞400 18 15 124 Vildagliptin (Norvatis) ^b >10 62 >10 1.3 Sitagliptin (Merck) ^c ＞100 18 48 ＞100 Saxagliptin (BMS) ＞400 13 0.17 0.061

a. See WO 06/104356 assigned to the applicant

b. Data released in IDF in 2006

c. Kim, D., et al., J. Med Chem, 2005, 48, 141-151

As can be seen from the results in Table 2, the compounds of the present invention exhibit excellent DPP-IV selectivity compared with the DPP-IV inhibitors in WO 06/104356. These results demonstrate that the potential toxicity of the compounds due to side effects can be minimized as previously described.

Industrial Applicability

From the foregoing it is evident that the novel compounds of the present invention inhibit the activity of DPP-IV, thereby leading to an increase in insulin secretion and thus a reduction in blood glucose levels. Therefore, these compounds can be used for the treatment or prevention of diseases related to DPP-IV, such as diabetes (especially type II), obesity and the like.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various Modifications, additions and substitutions are all possible.

Claims (12)

1. A compound represented by formula 1 or a pharmaceutically acceptable salt thereof:

in A is selected from: substituents of formula 2:

in R ₁ is hydrogen or CF ₃ , R ₂ is selected from: hydrogen, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₃ -C ₁₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₁₀ heterocycloalkyl, substituted or unsubstituted C ₄ -C ₈ aryl and substituted or unsubstituted C ₃ -C ₇ heteroaryl; and Substituents of formula 3:

Wherein R ₃ is selected from: hydrogen, halogen and substituted or unsubstituted C ₁ -C ₄ alkyl; B is selected from: substituents of formula 4:

in X is -CR ₄ R ₅ -or -CO-, wherein R ₄ and R ₅ are each independently hydrogen or hydroxyl, with the proviso that at least one of _R and _R is hydroxyl, R ₆ , R ₇ , R ₈ and R ₉ are each independently selected from: hydrogen, halogen, and substituted or unsubstituted C ₁ -C ₄ alkyl, n is 0 or 1; Substituents of Formula 5:

in X is -CR ₁₀ R ₁₁ -or -CO-, wherein R ₁₀ and R ₁₁ are each independently hydrogen or hydroxyl, with the proviso that at least one of R ₁₀ and R ₁₁ is hydroxyl, R ₁₂ , R ₁₃ and R ₁₄ are each independently selected from hydrogen, halogen, and substituted or unsubstituted C ₁ -C ₄ alkyl, Y is oxygen or sulfur; Substituents of formula 6:

in X is -CR ₁₅ R ₁₆ -or -CO-, wherein R ₁₅ and R ₁₆ are each independently hydrogen or hydroxyl, with the proviso that at least one of R ₁₅ and R ₁₆ is hydroxyl, Z is -CH- or oxygen, with the proviso that when Z is oxygen, R ₁₉ is absent, R ₁₇ , R ₁₈ and R ₁₉ are each independently selected from: hydrogen, halogen, and substituted or unsubstituted C ₁ -C ₄ alkyl; and A substituent of formula 7;

in X is -CR ₂₀ R ₂₁ -or -CO-, wherein R ₂₀ and R ₂₁ are each independently hydrogen or hydroxyl, with the proviso that at least one of R ₂₀ and R ₂₁ is hydroxyl, R ₂₂ is substituted or unsubstituted C ₁ -C ₄ alkyl. 2. The compound of claim 1, wherein when the alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is substituted, the substituent is _C1 - _C10 alkyl or halogen. 3. The compound of claim 2, wherein the halogen is fluorine. 4. The compound of claim 1, wherein A in Formula 1 is a substituent represented by Formula 2, wherein R ₁ is hydrogen or CF ₃ , R ₂ is selected from: hydrogen, and C ₁ -C ₅ alkyl , C ₃ -C ₆ cycloalkyl, C ₃ -C ₁₀ heterocycloalkyl, C ₄ -C ₈ aryl and C ₃ -C ₇ heteroaryl, each of which is optionally substituted by halogen. 5. The compound of claim 4, wherein when _R is unsubstituted or halogen substituted C ₃ -C ₁₀ heterocycloalkyl, or unsubstituted or halogen substituted C ₃ -C ₇ heteroaryl, the Said heterocycloalkyl or heteroaryl is any one selected from the following: furan, thiophene, pyrrole, pyrrolidine, imidazole, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazole morphine, thiazole, thiazoline, isothiazole, isothiazolidine, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazole Alkane, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, pyridine, pyridone, pyridazine, pyrazine, pyrimidine, piperidine, piperazine, morpholine, pyridazinone, tetrazole , triazoles, triazolidines and azapines. 6. The compound of claim 4, wherein R _is selected from: trifluoromethyl, propyl, butyl, t-butyl, cyclobutyl, pyridine, furan, methoxyethyl, thiophene and 4- Fluorophenyl. 7. The compound of claim 1, wherein B in Formula 1 is a substituent represented by Formula 4, wherein X is -(CH-OH)- or -CO-, R _{and R are} each independently selected From hydrogen, fluorine and unsubstituted C ₁ -C ₄ alkyl, R ₈ and R ₉ are each independently hydrogen. 8. The compound of claim 1, wherein the compound is a compound of formula 1a:

wherein A and B are as defined in formula 1. 9. The compound of claim 1, wherein the compound is any one of the following compounds: 1-[(2S)-amino-4-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-4- Oxygen-butyl]-5,5-difluoro-(6R)-hydroxyl-piperidin-2-one, 1-[(2S)-amino-4-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)-4- Oxygen-butyl]-5,5-difluoro-(6S)-hydroxy-piperidin-2-one, 1-[(S)-2-amino-4-oxo-4-(2-propyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine- 7-yl)-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-amino-4-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl )-4-oxo-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-amino-4-(2-cyclobutyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl )-4-oxo-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-amino-4-oxo-4-(2-pyridin-4-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-amino-4-oxo-4-(2-pyridin-4-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-amino-4-(2-furan-2-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine-7 -yl)-butyl]-4-oxo-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one, 1-[(S)-2-amino-4-(2-furan-2-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine-7 -yl)-butyl]-4-oxo-butyl]-5,5-difluoro-6-hydroxy-piperidin-2-one, 1-{(S)-2-amino-4-[2-(2-methoxy-ethyl)-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4- d] pyrimidin-7-yl]-4-oxo-butyl}-5,5-difluoro-6-hydroxyl-piperidin-2-one, 1-{(S)-2-amino-4-[2-(2-methoxy-ethyl)-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4- d] pyrimidin-7-yl]-4-oxo-butyl}-5,5-difluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-Amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-Amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5,5-difluoro-6-hydroxyl-piperidin-2-one, 1-{(S)-2-amino-4-[2-(4-fluoro-phenyl)-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d] Pyrimidin-7-yl]-4-oxo-butyl}-5,5-difluoro-6-hydroxy-piperidin-2-one, 1-[(S)-2-amino-4-(2,4-bis-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl)- 4-oxo-butyl]-3,3-difluoro-piperidine-2,6-dione, 1-[(S)-2-amino-4-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl )-4-oxo-butyl]-5-fluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-amino-4-(2-furan-2-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine-7 -yl)-4-oxo-butyl]-5-fluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-Amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5-fluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-amino-4-oxo-4-(2-propyl-5-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidine- 7-yl)-butyl]-5-fluoro-6-hydroxy-piperidin-2-one, 1-[(S)-2-amino-4-oxo-4-(2-pyridin-4-yl-5-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-5-fluoro-6-hydroxyl-piperidin-2-one, 1-[(S)-2-Amino-4-oxo-4-(2-thiophen-3-yl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d ]pyrimidin-7-yl)-butyl]-3-methyl-piperidine-2,6-dione, and 1-[(S)-2-amino-4-(2-tert-butyl-4-trifluoromethyl-5,8-dihydro-6H-pyrido[3,4-d]pyrimidin-7-yl )-4-oxo-butyl]-3-methyl-pyrrolidine-2,6-dione. 10. A pharmaceutical composition for inhibiting dipeptidyl peptidase-IV (DPP-IV), comprising the compound of formula 1 according to claim 1 or a pharmaceutically acceptable salt thereof. 11. The composition according to claim 10, wherein the pharmaceutical composition is used for the treatment or prevention of diseases associated with DPP-IV. 12. The composition of claim 11, wherein the disease associated with DPP-IV is diabetes or obesity.